Identification Of WRKY Transcription Factor Family And Functional Analysis In Tea Plant

Tea plant(Camellia sinensis),an important,economic woody plant,is known for its health benefits.Being an economic crop,its growth is limited by many environmental conditions,including disease and abiotic stresses.Increasing studies have demonstrated that WRKYs and VQ-motif proteins play pivotal roles in plant growth and abiotic stresses.Here,9 Camellia sinensis WRKY transcription fators(CsWRKYs)genes and 15 VQ motif-containing proteins(Cs VQs)were identified from tea cultivar ‘Longjing 43',and their expression profiles were investigated in different tissues under various abiotic stresses.Meanwhile,the members of CsWRKYs and Cs VQs gene family and their interactions were analyzed based on genomic data in ‘Shuchazao'.In order to investigate whether CsWRKY TFs participate in plant growth and respond to stress treatment,CsWRKYs genes were heterogeneous expressed both in Arabidopsis and tobacco.These results provide a theoretical basis for the response mechanism of CsWRKY and Cs VQ genes in plant growth and stress tolerance.The main findings are as follows:(1)Identification and expression analysis of CsWRKYs genes in tea plant.9 CsWRKYs were cloned by RT-PCR,and bioinformatic information including relative molecular mass,theoretical isoelectric point,transmembrane domain and signal peptide of these sequences were predicted.Tissue-specific expression analysis suggested that the expression levels of most CsWRKYs were relatively lower in flowers and stems than in other tissues.Among them,most CsWRKYs were significantly higher in leaves.The expression profiles of 9 CsWRKYs in leaves and roots were varied when exposed to diverse abiotic stresses,in which majority of CsWRKYs were significantly induced.These results indicated that CsWRKYs genes were widely involved in development process as well as plant abiotic stress responses.(2)Identification of 15 VQ motif-containing proteins in tea plant,and they could be divided into 7 groups(groups I,II,V,VI,VIII,IX and X)with 3,3,1,3,1,2 and 2 members in each group,respectively.Tissue-specific expression profiles of Cs VQs were similar to those of CsWRKYs genes: most genes have a higher abundance in leaves.When exposed to different abiotic stresses,majority of these Cs VQs genes were remarkably up-regulated.Variations in expression patterns were also found in leaves and roots.These results indicated that Cs VQs play important roles in the growth and development of tea plant as well as abiotic stress responses.(3)A total of 74 putative CsWRKYs and 30 putative Cs VQs were screened from genomic data in ‘Shuchazao'.The WRKYGQK core sequence in tea plant contains at least three variants,including WRKYGQK,WRKYGKK and WRKYGRK.There are 4(LTG,FTG,VTG and VTA)types of VQ-motif domains in tea plant.The protein-protein interactions among WRKY and VQ proteins were predicted based on their orthologous relationships in Arabidopsis and STRING website.Thirteen pairs of potential WRKY-VQ interacting proteins were obtained in tea plant.The majority of interacting proteins had similar tissue expression patterns,especially CsWRKY26/Cs VQ4,CsWRKY26/Cs VQ9,and CsWRKY57/Cs VQ23-1.(4)Functional evaluation of CsWRKY26 in abiotic resistance.CsWRKY26 protein was localized in the nucleus.CsWRKY26 could interact with Cs VQ4-1/-9-1/-15/-21/-23-1 by removing part of a N-terminal self-activated region in Yeast-two-hybridization systems.The expression patterns of CsWRKY26 were elevated under abiotic stresses and natural drought conditions.A group of elements which respond to such environmental stresses as light-inducible,plant growth-response,and stress-response cis-elements(MBS and W-box)were found in the CsWRKY26 promoter region.GUS staining revealed a similarity expression profile of CsWRKY26 compared to the results from our previous q RT-PCR analysis.CsWRKY26-overexpressing Arabidopsis lines were not sensitive to ABA treatment,and exhibit increased resistance to salt stress caused by Na Cl,and osmotic stress induced by polyethylene glycol(PEG),and mannitol during the germination process.Meanwhile,CsWRKY26-overexpressing Arabidopsis plants showed greater resistance to exogenous ABA and PEG treatments compared to WT according to seedling growth assay.The expression levels of some stress-tolerant genes such as RD22,NCED3,and P5CS1(which is a key enzyme in proline synthesis)were upregulated in transgenic lines copared to WT when exposed to 15% PEG simulated drought stress conditions.Under natural drought stress,the growth performance of transgenic lines was better than that of WT,concurrent with significantly lower EL and higher proline content.RNA-seq analysis further showed that 1700 genes were differentially expressed,including 559 specific up-regulated genes and 428 specific down-regulated genes.Among these up-regulated genes,SWEET16 and SUS25,two genes involved in glucose metabolism,were both up-regulated under drought condition.In addition,multiple transcription factors such as b HLH(b HLH30/-71/-72/-93/-137),MYB(MYB24/-29),and ERF(ERF008/-010/-034/-042)as well as auxin response proteins such as IAA27/-29,and stress response gene RD22 were also up-regulated.These findings indicated that CsWRKY26 enhance plant drought tolerance might through reducing the degree of cell membrane injury,increasing osmotic substances content,and promoting the expression levels of stress-related genes,as well as activating some signaling pathways such as ABA,ethylene and auxin.(5)Function explorations of the CsWRKY7 and CsWRKY12 TFs in tea plant.Both CsWRKY7 and CsWRKY12 proteins contained nuclear localization signals.Two length of 1680 bp and 1835 bp 5'-flanking sequences of CsWRKY7 and CsWRKY12 were cloned from the leaves of ‘Longjing 43',respectively,and both sequences contained multiple lightresponse,plant growth-response and stress-response cis-elements.Tobacco plants overexpressing CsWRKY7 and CsWRKY12 were obtained according to the leaf disc method,which provided materials for subsequent experiments.Seed germination and root growth assay indicated that overexpressed CsWRKY7 in transgenic Arabidopsis was not sensitive to Na Cl,mannitol,PEG6000,and low concentration of ABA treatments.CsWRKY7 overexpressing Arabidopsis showed a late-flowering phenotype under normal conditions compared to wild type.Furthermore,gene expression analysis showed that the transcription levels of the flowering time integrator gene FLOWERING LOCUS T(FT),the floral meristem identity genes APETALA1(AP1)and LEAFY(LFY)were lower in WRKY7-OE than in the WT,indicating that CsWRKY7 delay flowering time might through inhibiting the transcription level of AP1 and LFY.CsWRKY12-overexpressing plants promoted flowering under short day time(SD).Furetermore,overexpression of CsWRKY12 could restore the late-flowering phenotype of wrky12 mutant under SD.The length of stem in transgenic lines were higher than that in WT,and transgenic plants have more stem leaves.In restored lines,the number of stem leaves,siliques,and stem length were significantly higher than that of the mutant.The expression of flowering-related genes in transgenic lines were analyzed by q RT-PCR,which demonstrated that three genes,FT,AP1 and LFY,were up-regulated in transgenic Arabidopsis compare to WT.These findings indicated that CsWRKY12 promote flowering time might through directly or indirectly regulating the transcription level of FLY,FT and AP1.